Abstract

There is a general necessity to improve the specificity and efficiency of the polymerase chain reaction (PCR), and exploring the PCR-enhancing mechanism still remains a great challenge. In this paper we report the use of branched polyethyleneimine (PEI)-based derivatives and hybrid nanocomposites as a novel class of enhancers to improve the specificity and efficiency of a nonspecific PCR system. We show that the surface-charge polarity of PEI and PEI derivatives plays a major role in their effectiveness to enhance the PCR. Positively charged amine-terminated pristine PEI, partially (50%) acetylated PEI (PEI-Ac(50)), and completely acetylated PEI (PEI-Ac) are able to improve PCR efficiency and specificity with an optimum concentration order of PEI < PEI-Ac(50) < PEI-Ac, whereas negatively charged carboxyl-terminated PEI (PEI-SAH; SAH denotes succinamic acid groups) and neutralized PEI modified with both polyethylene glycol (PEG) and acetyl (Ac) groups (PEI-PEG-Ac) are unable to improve PCR specificity and efficiency even at concentrations three orders of magnitude higher than that of PEI. Our data clearly suggests that the PCR-enhancing effect is primarily based on the interaction between the PCR components and the PEI derivatives, where electrostatic interaction plays a major role in concentrating the PCR components locally on the backbones of the branched PEI. In addition, multiwalled carbon nanotubes modified with PEI and PEI-stabilized gold nanoparticles are also able to improve the PCR specificity and efficiency with an optimum PEI concentration less than that of the PEI alone, indicating that the inorganic component of the nanocomposites may help improve the interaction between PEI and the PCR components. The developed PEI-based derivatives or nanocomposites may be used as efficient additives to enhance other PCR systems for different biomedical applications.

The effects of PEI-based derivatives on the nonspecific polymerase chain reaction (PCR) system. Lane M is the DL 2000 marker, and the rightmost lane in each panel is the negative control amplified without template and additives. (A) PEI was added into the PCR mixture. From lane 1 to 4, its final concentration is 0, 20, 40, 60 μg/L, respectively. (B) PEI-Ac50 was added into the PCR mixture. From lane 1 to line 5, its final concentration is 0, 45, 90, 112, and 135 μg/L, respectively. (C) PEI-Ac was added into the PCR mixture. From lane 1 to 4, its final concentration is 0, 36, 360, and 720 μg/L, respectively. (D) PEI-SAH was added into the PCR mixture. From lane 1 to 7, its final concentration is 0, 17, 34, 102, 170, 238, and 340 mg/L, respectively. (E) PEI-PEG-Ac was added into the PCR mixture. From lane 1 to 7, its final concentration is 0, 16.4, 32.8, 65.6, 84, 131, and 164 mg/L, respectively.Abbreviations: PEI, polyethyleneimine; PEI-Ac50, partially (50%) acetylated PEI; PEI-Ac, completely acetylated PEI; PEI-SAH, negatively charged carboxyl-terminated PEI (SAH denotes succinamic acid groups); PEI-PEG-Ac, neutralized PEI modified with both polyethylene glycol and acetyl groups.

The effects of MWCNTs and MWCNT/PEI on the specificity of the nonspecific polymerase chain reaction system. Lane M is the DL 2000 marker, and the rightmost lane is negative control in each panel. (A) MWCNT was added into the polymerase chain reaction mixture. From lane 1 to 4, its final concentration is 0, 3.1, 6.2, and 12.4 mg/L, respectively. (B) MWCNT-PEI was added into the polymerase chain reaction mixture. From lane 1 to 5, its final concentration is 0, 23.6, 47.2, 94.2, and 188.8 μg/L, respectively.Abbreviations: MWCNTs, multiwalled carbon nanotubes; MWCNT/PEI, polyethyleneimine-modified multiwalled carbon nanotubes.

The effect of the polyethyleneimine-stabilized gold nanoparticles on the nonspecific polymerase chain reaction system. Lane M is the DL 2000 marker, and the rightmost lane is the negative control. From lane 1 to 5, the final polyethyleneimine concentration is 0, 12.5, 25, 36, and 75 μg/L, respectively.